Köksal Elif Senem, Belletati Patrícia F, Reint Ganna, Olsson Ragni, Leitl Kira D, Kantarci Ilayda, Gözen Irep
Centre for Molecular Medicine Norway, Faculty of Medicine, University of Oslo.
Centre for Molecular Medicine Norway, Faculty of Medicine, University of Oslo; Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Oslo; Department of Chemistry and Chemical Engineering, Chalmers University of Technology;
J Vis Exp. 2019 Jan 22(143). doi: 10.3791/58923.
We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca in the ambient buffer. Subsequent depletion of Ca by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.
